EP0186551B1 - Medicament containing as a mixture at least one immunotoxin and at least one polymer containing mannose - Google Patents

Medicament containing as a mixture at least one immunotoxin and at least one polymer containing mannose Download PDF

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EP0186551B1
EP0186551B1 EP85402319A EP85402319A EP0186551B1 EP 0186551 B1 EP0186551 B1 EP 0186551B1 EP 85402319 A EP85402319 A EP 85402319A EP 85402319 A EP85402319 A EP 85402319A EP 0186551 B1 EP0186551 B1 EP 0186551B1
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Prior art keywords
immunotoxin
mannan
chain
antibody
mannose
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EP0186551A1 (en
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Pierre Casellas
Bernard Rés. du Plan des 4 Seigneurs Bourrie
Pierre Gros
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Sanofi SA
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Sanofi SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit

Definitions

  • the therapeutic effects of activated or not activated immunotoxins can only be fully manifested insofar as the immunotoxin can, by its antibody part, localize in vivo, in active form, on the cell target to be destroyed (condition sine qua non any expression of immunotoxin activity).
  • the ability of the immunotoxin to locate on the target depends primarily on the ability of the immunotoxin to remain in the bloodstream and the extracellular fluids in active form for sufficient time to reach its target. cell target and at concentrations high enough for the occupancy rate of the corresponding antigenic sites to be high.
  • the Applicant has carried out numerous studies which have made it possible to establish the kinetics of plasma elimination of immunotoxins after intravenous injection in various animal models. It appeared that, after injection, the plasma level of biologically active immunotoxin decreases very quickly and very significantly. Thus, typically, in the rabbit, in a model using an immunotoxin constructed by coupling, using an arm with a disulfide bridge, the ricin A chain to a monoclonal antibody directed against the T65 antigen of human T lymphocytes, it appears that 97% of the immunotoxin present in the blood circulating at time 0 of the injection disappears in 30 min and 99.9% in 17 h.
  • the present invention relates to new drugs making it possible to inhibit the rapid elimination of immunotoxins from the plasma after injection, without the intrinsic properties characteristic of immunotoxins being affected.
  • the medicaments according to the invention packaged so as to be used by injection, preferably by intravenous route, comprise, in combination, at least one immunotoxin obtained from. toxin or toxic subunit or fragment of natural or semi-synthetic or synthetic toxic subunit comprising polysaccharide groups containing mannose residues, in particular in the terminal position, whatever the constitutive antibody and whatever the type of bond chosen to link the antibody to the toxin or toxic subunit or toxic fragment, and at least one carbohydrate, polysaccharide or polysaccharide polymer with an average molecular weight greater than 1000 comprising from 20 to 100% of mannose residues, whatever the type of chain linking these mannose residues to each other or to other sugars.
  • mannans represent a type of substance which is particularly advantageous for increasing the plasma levels of immunotoxins.
  • the term mannan is used here to designate any carbohydrate polymer, polysaccharide or polysaccharide, of average molecular weight greater than 1000 comprising a significant proportion of mannose residues and more particularly from 20 to 100% of mannose residues, whatever the type of osidic sequence connecting these mannose residues to each other or to other sugars.
  • natural mannans isolated from yeasts for example Saccharomyces cerevisiae.
  • the protein-mannan complex is a mixture of macromolecules in which the polysaccharide component represents 50 to 90% of the complex.
  • the mannan fraction is itself a polymer of D-mannose. It is made up of an armature of mannose residues coupled in position a 1 6 6 to which are added side chains of different lengths comprising ⁇ 1 ⁇ 3 and ⁇ 1 ⁇ 2 links.
  • mannan used at doses where it does not present in itself, nor in combination with the immunotoxin any toxicity for the animal, makes it possible to increase in an extremely important way (by a factor of the order of 100) and lasting the plasma concentration of immunotoxins, which considerably improves their localization on the target and avoids binding inhibition phenomena due to the presence of free antibodies in the preparations, as indicated above .
  • glycoproteins recognized by these receptors - present essentially on Kupffer's cells - are rapidly eliminated from the blood circulation by fixation on these cells which metabolize them. This is well documented in particular in the case of ⁇ -glucuronidase, as well as in the case of ribonuclease B (Arch. Biochem. Biophys. (1978) 188, 418; Advances in Enzymology Meister A. Ed New York (1974); Pediat. Res. (1977) 11, 816).
  • mannanes to inhibit the rapid plasma elimination of ricin A chain immunotoxins applies equally, for the reasons indicated above, to the uncoupled ricin A chain or to any hybrid, natural or semi-molecule synthetic, containing the ricin A chain and, in particular, to all the immunotoxins containing the ricin A chain, whatever the type of binding chosen to link the antibody to the ricin A chain.
  • This property of mannans applies more generally for the same reasons to any immunotoxin, whatever the toxin used to produce it, if this toxin or toxic subunit contains polysaccharide groups containing mannose residues, in particular in the terminal position , whatever the constitutive antibody and whatever the type of binding chosen to bind the antibody to the toxin or toxic subunit.
  • the purpose of this example is to demonstrate the modifications in the kinetics of elimination of the immunotoxins (as well as those of their constituent elements) in the presence or in the absence of mannan.
  • the conjugate called IT-T101 is obtained by reaction between an antibody against human T cells (antibody T101 directed against the T65 antigen) substituted by an activated disulfide group and the ricin A chain.
  • the preparation and the cytotoxic properties of this conjugate have been described in French patent application No. 8121836 of the applicant.
  • the tT-T101 conjugate is administered to rabbits by a single injection into an ear vein.
  • the amount injected corresponds to 1.25 mg of immunotoxin per kg of body weight, ie 0.415 mg / kg expressed in chain A, and 0.835 mg / kg expressed in antibody.
  • Blood samples are taken over time on heparin.
  • the plasmas are analyzed using an immunoradiometric test below designated by the abbreviation IRM-1.
  • This technique has the advantage of assaying the immunotoxin without modifying it.
  • This assay is carried out in microtiter plates (for example: “NUNC-TSP screening system” Poly Labo Block, France), the cover of which is provided with hyper-absorbent tips which plunge into the wells of the base. These points constitute the solid phases.
  • Ricin antichain A sheep antibodies hereinafter referred to as Acl
  • Acl purified by affinity chromatography, are adsorbed on the phases solid. For this, 200 ⁇ l of an Acl solution at 10 ⁇ g / ml in the 20 mM phosphate buffer pH 7, 150 mM NaCi are distributed in the wells.
  • the tips are brought into contact first with the Acl solution for 24 h at 4 ° C and then with fetal calf serum for 3 h at 20 ° C to saturate all the binding sites.
  • the saturated immunoabsorbent is then brought into contact for 3 h at 20 ° C with the plasma samples at different dilutions or with IT-T101 immunotoxin solutions of known concentrations for establishing the calibration curve. Washing is carried out with 20 mM phosphate buffer, pH 7, 150 mM NaCl, then the immunoabsorbent is brought into contact for 2 h at 20 ° C. with goat anti-mouse IgG antibodies purified by affinity chromatography and radiolabelled ( hereinafter designated by the abbreviation Ac2).
  • Radiolabelling of Ac2 is carried out with iodine 125 in the presence of chloramine T according to the method of Greenwood and Hunter (Biochem. J., (1963) 89, 114); the specific activity of radiolabelled Ac2 is 5 to 10 ⁇ Ci / mg. 10 6 cpm of radiolabelled Ac2 are introduced under 200 ml in a 20 mM phosphate buffer pH 7, 150 mM NaCl, 0.1% bovine serum albumin. After washing, in a 20 mM phosphate buffer pH 7, 150 mM NaCl, the tips are detached and the amount of bound Ac2 is measured by counting the radioactivity. The measurement of the immunotoxin concentration in the samples to be assayed is made by reference to the calibration curve carried out with the IT-T101 introduced at different known concentrations.
  • This antibody was prepared and purified as indicated in French patent application No. 8121836.
  • the T101 antibody is injected into rabbits intravenously at a dose of 0.835 mg / kg. Plasma samples are taken as before.
  • the antibody concentration in the samples is measured by radioimmunometric assay (MRI-2). This test is carried out under the same conditions as the MRI-1 test, except that the Acl solution is here a 10 mg / ml solution of goat anti-mouse IgG antibodies purified by affinity chromatography.
  • the Ac2 antibody is identical to that of the MRI-1 test.
  • the measurement of the concentration of T101 antibody in the samples to be assayed is made by reference to the calibration curve carried out with the T101 antibody introduced at different known concentrations.
  • the ricin chain A was prepared and purified as indicated in prior French patents No. 78/27838 and addition No. 79/24655.
  • the A chain is injected into the rabbit intravenously at a dose of 0.415 mg / kg. Plasma samples are taken as before.
  • the concentration of chain A in the samples is measured by immunoradiometric assay (MRI-3). This test is carried out under the same conditions as the MRI-1 test, the antibodies Ad, absorbed on the solid phase, also being sheep antibodies against ricin antichain A purified by affinity chromatography and the antibodies Ac2 being the same radiolabeled antibodies as described in IRM-1.
  • the measurement of the ricin A chain concentration in the samples to be assayed is made by reference to a calibration curve carried out with the ricin A chain introduced at different known concentrations.
  • the detection threshold for these three products is 1 ng / ml.
  • the study of intra- and inter-test reproducibility gives coefficients of variation of less than 10% for the concentration values in the range from 1 to 200 ng / ml.
  • Figure 1 shows the plasma elimination curve of IT-T101 which has two phases (curve 1). In the first phase, the product disappears quickly (around 97% in 30 min); in the second phase, the decrease is slower. The first elimination phase observed with IT-T101 does not appear in the elimination kinetics of the T101 antibody where only a slow elimination phase is recorded (curve 2). On the other hand, the kinetics of plasma elimination from the uncoupled A chain is very comparable to that of IT-T101: 1 h after the injection, only 0.7% of the administered dose remains in the plasma. (curve 3).
  • Figure 2 shows the plasma elimination curve as a function of time of IT-T101 injected intravenously in association with mannan at the total dose of 0.416 g / kg (curve 2).
  • the first phase of elimination - responsible for the disappearance of most of the product - is practically abolished, which leads to a major increase in the level of active plasma immunotoxin.
  • the concentration of IT-Ti 01 is 100 times higher, when the immunotoxin has been combined with mannan, than it is in the absence of mannan (curve 1).
  • This example demonstrates the hepatic uptake of the ricin A chain after intravenous injection and the inhibition of this uptake by mannan.
  • the i-125 radiolabelled A chain is injected into Charles River France CD1 mice intravenously in the absence or presence of mannan at a dose of 1 g / kg.
  • two animals are anesthetized.
  • the abdominal cavity is open, the vena cava sectioned, the liver is washed with 10 ml of physiological water by injection into the portal vein.
  • the entire liver is removed and the radioactivity is determined.
  • the results are represented as a percentage of the number of cpm fixed on the liver relative to the number of total cpm injected (FIG. 5).
  • the ricin A chain is very early and efficiently picked up by the liver as indicated by the peak of radioactivity.
  • the cytotoxicity was evaluated by measuring the incorporation of 14C-leucine by the target cells (CEM cells) after 24 h of incubation at 37 ° C. in the presence of known concentrations of the immunotoxin studied, or of reference cytotoxic substances, in the absence or presence of mannan at a concentration of 10 mg / ml.
  • the technique used is that previously described (J. Biol. Chem. 1984, 259 (15), 9359).
  • the immunotoxin alone or in its form activated by ammonium chloride fully retains its activity. Likewise, the intrinsic toxicity of the A chain is not modified. So in. presence of mannan, the characteristic cytotoxicity properties of the immunotoxin are not altered.
  • the purpose of this example is to demonstrate the effect of mannan on the action of an immunotoxin in an "in vivo" test.
  • the experiment was carried out in BL 1.1 (Thy 1.2 negative) mice [International Journal of Cancer 24, 168-177, (1979)].
  • the immunotoxin used is the conjugate combining via a disulfide bond the anti-Thy 1.2 antibody (AT15E antibody) and the A chain of Ricin and prepared according to the process described in the French patent applications in the name of the applicant and cited above.
  • mice receive 0.5 x 10 4 T2 cells per day intravenous injection (Thy 1.2 positive murine lymphoma cells) and are randomized before treatment.
  • the animals are observed for 50 days and mortality is noted.
  • Figure 6 gives the percentage of surviving animals as a function of the time elapsed after treatment.
  • Curve 1 concerns the animals which received the immunotoxin alone and curve 2 the animals which received the immunotoxin plus mannan combination.
  • the combination constituted by an immunotoxin and mannan can be used for the treatment of conditions, cancerous or not, whose target cells are recognized by the antibody used for the preparation of the immunotoxin.
  • the treatment should be carried out with a sufficient dose of immunotoxin associated with an amount of mannan which can vary from 10 mg to 1 g / kg each time the immunotoxin is administered.
  • the optimal methods of administration of the constituents of the association as well as the duration of treatment must be determined in each case depending on the subject and the nature of the condition to be treated.
  • the new drugs according to the invention are packaged for use by the injectable route and, preferably, by the intravenous route.
  • the constituents of the combination will be kept separate and may be mixed if necessary only at the time of use, in the syringe or the perfusion solvent.

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Abstract

The invention relates to pharmaceutical compositions comprising a combination of at least one immunotoxin and at least one mannose-containing polymer. This combination makes it possible to inhibit the rapid elimination of immunotoxins from the plasma after injection.

Description

Dans les brevets français antérieurs n° 78/27838 et addition n° 79/24665 et dans les demandes de brevets français n° 81/07596 et n° 81/21836, la demanderesse a décrit la préparation de produits anticancéreux dits conjugués, obtenus par couplage, par liaison covalente, de la chaîne A de ricine avec des anticorps ou fragments d'anticorps dirigés contre un antigène porté par la cellule à détruire. Les produits de ce type sont désignés dans la présente demande sous le nom générique d'Immunotoxines.In prior French patents No. 78/27838 and addition No. 79/24665 and in French patent applications No. 81/07596 and No. 81/21836, the Applicant has described the preparation of so-called conjugated anticancer products obtained by coupling, by covalent bond, of the ricin A chain with antibodies or fragments of antibodies directed against an antigen carried by the cell to be destroyed. Products of this type are designated in the present application under the generic name of Immunotoxins.

Dans ses demandes de brevets français n° 81/21836, n° 82/02091, n° 82/04119, n° 82/04047 et n° 82/04547, la demanderesse a également montré la capacité de certaines substances (sels d'ammonium, ionophores carboxyliques monovalents, méthylamine, chloroquine et conjugués enzymatiques susceptibles de libérer de l'ammoniac) de potentialiser l'action cytotoxique des immunotoxines.In its French patent applications n ° 81/21836, n ° 82/02091, n ° 82/04119, n ° 82/04047 and n ° 82/04547, the plaintiff also showed the capacity of certain substances (salts of ammonium, monovalent carboxylic ionophores, methylamine, chloroquine and enzymatic conjugates capable of releasing ammonia) to potentiate the cytotoxic action of immunotoxins.

Toutefois, les effets thérapeutiques des immunotoxines activées ou non ne peuvent se manifester pleinement que dans la mesure où l'immunotoxine peut, par sa partie anticorps, se localiser in vivo, sous forme active, sur la cible cellulaire à détruire (condition sine qua non à toute expression d'activité des immunotoxines). La capacité de l'immunotoxine à se localiser sur la cible dépend en tout premier lieu de l'aptitude de l'immunotoxine à demeurer dans la circulation sanguine et les fluides extra-cellulaires sous forme active pendant des temps suffisants pour qu'elle atteigne sa cible cellulaire et à des concentrations suffisamment fortes pour que le taux d'occupation des sites antigéniques correspondants soit élevé.However, the therapeutic effects of activated or not activated immunotoxins can only be fully manifested insofar as the immunotoxin can, by its antibody part, localize in vivo, in active form, on the cell target to be destroyed (condition sine qua non any expression of immunotoxin activity). The ability of the immunotoxin to locate on the target depends primarily on the ability of the immunotoxin to remain in the bloodstream and the extracellular fluids in active form for sufficient time to reach its target. cell target and at concentrations high enough for the occupancy rate of the corresponding antigenic sites to be high.

La demanderesse a effectué de nombreuses études qui ont permis d'établir la cinétique d'élimination plasmatique des immunotoxines après injection intraveineuse dans différents modèles animaux. Il est apparu que, après l'injection, le taux plasmatique d'immunotoxine biologiquement active décroît très rapidement et de façon très importante. Ainsi, typiquement, chez le lapin, dans un modèle utilisant une immunotoxine construite en couplant à l'aide d'un bras à pont disulfure la chaîne A de ricine à un anticorps monoclonal dirigé contre l'antigène T65 des lymphocytes T humains, il apparaît que 97 % de l'immunotoxine présente dans le sang circulant au temps 0 de l'injection disparaissent en 30 min et 99,9 % en 17 h. Les résultats obtenus sont analogues si le bras reliant l'anticorps à la chaîne A de ricine comporte une liaison thioéther au lieu d'une liaison disulfure. Cette rapide disparition de l'immunotoxine est bien évidemment préjudiciable à l'expression de sa capacité cytotoxique complète, en empêchant que l'immunotoxine ne sature de façon durable une proportion élevée des antigènes cibles portés par les cellules à détruire. En outre, la comparaison des cinétiques d'élimination plasmatique des immunotoxines avec celles des anticorps non conjugués correspondants montre, à l'inverse, que les anticorps - comme cela èst bien connu - se maintiennent dans le plasma à un haut niveau pendant des temps relativement longs. Or, il existe toujours, même dans les préparations d'immunotoxines les plus purifiées, un certain taux résiduel d'anticorps non conjugués. Par le jeu des vitesses différentielles d'élimination des immunotoxines et des anticorps, progressivement les anticorps non conjugués, initialement très minoritaires, deviennent majoritaires au bout de quelques heures et donc, progressivement, ces anticorps deviennent par compétition de puissants antagonistes pour la fixation des immunotoxines sur leurs cibles.The Applicant has carried out numerous studies which have made it possible to establish the kinetics of plasma elimination of immunotoxins after intravenous injection in various animal models. It appeared that, after injection, the plasma level of biologically active immunotoxin decreases very quickly and very significantly. Thus, typically, in the rabbit, in a model using an immunotoxin constructed by coupling, using an arm with a disulfide bridge, the ricin A chain to a monoclonal antibody directed against the T65 antigen of human T lymphocytes, it appears that 97% of the immunotoxin present in the blood circulating at time 0 of the injection disappears in 30 min and 99.9% in 17 h. The results obtained are similar if the arm connecting the antibody to the ricin A chain comprises a thioether bond instead of a disulfide bond. This rapid disappearance of the immunotoxin is obviously detrimental to the expression of its complete cytotoxic capacity, by preventing the immunotoxin from permanently saturating a high proportion of the target antigens carried by the cells to be destroyed. Furthermore, the comparison of the kinetics of plasma elimination of immunotoxins with those of the corresponding unconjugated antibodies shows, conversely, that the antibodies - as is well known - remain in the plasma at a high level for relatively long times. long. However, there is always, even in the most purified immunotoxin preparations, a certain residual level of unconjugated antibodies. By the play of differential rates of elimination of immunotoxins and antibodies, gradually the non-conjugated antibodies, initially very minority, become the majority after a few hours and therefore, gradually, these antibodies become by competition powerful antagonists for the binding of immunotoxins on their targets.

De ces études, apparaît clairement l'intérêt d'accroître la persistance plasmatique des immunotoxines, sous forme active, afin d'augmenter à la fois la durée et le taux d'occupation des antigènes-cibles et, par conséquent, d'améliorer les effets thérapeutiques des immunotoxines.From these studies, it appears clearly the advantage of increasing the plasma persistence of immunotoxins, in active form, in order to increase both the duration and the rate of occupation of the target antigens and, consequently, to improve the therapeutic effects of immunotoxins.

La présente invention a pour objet des nouveaux médicaments permettant d'inhiber l'élimination rapide des immunotoxines du plasma après injection, sans qu'il soit porté atteinte aux propriétés intrinsèques caractéristiques des immunotoxines.The present invention relates to new drugs making it possible to inhibit the rapid elimination of immunotoxins from the plasma after injection, without the intrinsic properties characteristic of immunotoxins being affected.

Les médicaments selon l'invention, conditionnés de façon à être utilisés par voie injectable, de préférence par voie intraveineuse, comportent, en association, au moins une immunotoxine obtenue à partir de. toxine ou sous-unité toxique ou fragment de sous-unité toxique naturelle ou semi-synthétique ou synthétique comportant des groupements polysaccharidiques contenant des résidus de mannose, en particulier en position terminale, quel que soit l'anticorps constitutif et quel que soit le type de liaison choisi pour relier l'anticorps à la toxine ou sous-unité toxique ou fragment toxique, et au moins un polymère glucidique, polyosidique ou polysaccharidique de poids moléculaie moyen supérieur à 1 000 comportant de 20 à 100 % de résidus mannose, quel que soit le type d'enchaînement reliant ces résidus mannose entre eux ou à d'autres sucres.The medicaments according to the invention, packaged so as to be used by injection, preferably by intravenous route, comprise, in combination, at least one immunotoxin obtained from. toxin or toxic subunit or fragment of natural or semi-synthetic or synthetic toxic subunit comprising polysaccharide groups containing mannose residues, in particular in the terminal position, whatever the constitutive antibody and whatever the type of bond chosen to link the antibody to the toxin or toxic subunit or toxic fragment, and at least one carbohydrate, polysaccharide or polysaccharide polymer with an average molecular weight greater than 1000 comprising from 20 to 100% of mannose residues, whatever the type of chain linking these mannose residues to each other or to other sugars.

D'une façon surprenante, il a été trouvé, selon la présente invention, que les mannanes représentent un type de substances particulièrement intéressantes pour accroître les taux plasmatiques des immunotoxines. Le terme de mannane est employé ici pour désigner tout polymère glucidique, polyoside ou polysaccharide, de poids moléculaire moyen supérieur à 1 000 comportant une proportion importante de résidus mannose et plus particulièrement de 20 à 100 % de résidus mannose, quel que soit le type d'enchaînement osidique reliant ces résidus mannose entre eux ou à d'autres sucres. En particulier et, à titre d'exemple non limitatif, on peut utiliser au sens de la présente invention des mannanes naturels isolés à partir de levures (par exemple Saccharomyces cerevisiae). Il s'agit alors de la fraction glucidique d'un peptidoglycane appartenant à la paroi cellulaire de ces levures. Le complexe protéine-mannane est un mélange de macromolécules dans lequel le composant polysaccharidique représente 50 à 90 % du complexe. La fraction mannane est elle-même un polymère de D-mannose. Elle est formée d'une armature de résidus mannose couplés en position a 1 6 6 à laquelle s'ajoutent des chaînes latérales de différentes longueurs comportant des liaisons α 1 → 3 et α 1 → 2.Surprisingly, it has been found, according to the present invention, that mannans represent a type of substance which is particularly advantageous for increasing the plasma levels of immunotoxins. The term mannan is used here to designate any carbohydrate polymer, polysaccharide or polysaccharide, of average molecular weight greater than 1000 comprising a significant proportion of mannose residues and more particularly from 20 to 100% of mannose residues, whatever the type of osidic sequence connecting these mannose residues to each other or to other sugars. In particular and, by way of nonlimiting example, it is possible to use, within the meaning of the present invention, natural mannans isolated from yeasts (for example Saccharomyces cerevisiae). It is then the carbohydrate fraction of a peptidoglycan belonging to the cell wall of these yeasts. The protein-mannan complex is a mixture of macromolecules in which the polysaccharide component represents 50 to 90% of the complex. The mannan fraction is itself a polymer of D-mannose. It is made up of an armature of mannose residues coupled in position a 1 6 6 to which are added side chains of different lengths comprising α 1 → 3 and α 1 → 2 links.

D'une façon surprenante, le mannane, utilisé à des doses où il ne présente ni en lui-même, ni en association avec l'immunotoxine aucune toxicité pour l'animal, permet d'accroître de façon extrêmement importante (d'un facteur de l'ordre de 100) et durable la concentration plasmatique des immunotoxines, ce qui améliore considérablement leur localisation sur la cible et évite des phénomènes d'inhibition de fixation dus à la présence d'anticorps libres dans les préparations, comme cela est indiqué précédemment.Surprisingly, mannan, used at doses where it does not present in itself, nor in combination with the immunotoxin any toxicity for the animal, makes it possible to increase in an extremely important way (by a factor of the order of 100) and lasting the plasma concentration of immunotoxins, which considerably improves their localization on the target and avoids binding inhibition phenomena due to the presence of free antibodies in the preparations, as indicated above .

L'absence remarquable de toxicité des mannanes en fait des substances de choix pour une utilisation pharmaceutique en association avec les immunotoxines. L'association de l'immunotoxine au mannane n'accroît pas de façon importante la toxicité propre de l'immunotoxine. Cette association n'interfère pas non plus avec les propriétés de cytotoxicité spécifique caractéristiques des immunotoxines en présence ou non des potentialisateurs déjà décrits.The remarkable lack of toxicity of mannans makes them the substances of choice for pharmaceutical use in combination with immunotoxins. The association of the immunotoxin with mannan does not significantly increase the inherent toxicity of the immunotoxin. This association also does not interfere with the specific cytotoxicity properties characteristic of immunotoxins in the presence or not of the potentiators already described.

Par ailleurs, des expériences de localisation in vivo de l'immunotoxine radiomarquée injectée chez des animaux sans cible spécifique ont montré que, dans les premières minutes après l'injection, le conjugué se localise préférentiellement dans le foie. Il en est de même pour la chaîne A qui suit le même devenir lorsqu'elle est injectée sous forme non couplée. Cela suggère fortement que l'immunotoxine se fixe dans le foie par l'intermédiaire de la chaîne A de ricine qu'elle contient. Il est connu que la chaîne A de ricine est une glycoprotéine donc les groupements polyosidiques comprennent des résidus de mannose et de N-acétylglucosamine, les résidus de mannose étant en position terminale (Agri. Biol. Chem. (1978) 42, 501). Il a été également établi l'existence au niveau du foie de récepteurs capables de reconnaître des glycoprotéines ayant de tels résidus de mannose terminaux.Furthermore, in vivo localization experiments of the radiolabelled immunotoxin injected into animals without a specific target have shown that, in the first minutes after injection, the conjugate preferentially localizes in the liver. It is the same for the chain A which follows the same fate when it is injected in uncoupled form. This strongly suggests that the immunotoxin binds to the liver through the ricin A chain it contains. It is known that the ricin A chain is a glycoprotein therefore the polysaccharide groups comprise residues of mannose and of N-acetylglucosamine, the residues of mannose being in the terminal position (Agri. Biol. Chem. (1978) 42, 501). It has also been established the existence in the liver of receptors capable of recognizing glycoproteins having such terminal mannose residues.

Il a été ainsi montré que les glycoprotéines reconnues par ces récepteurs - présents essentiellement sur les cellules de Kupffer - sont rapidement éliminées de la circulation sanguine par fixation sur ces cellules qui les métabolisent. Ceci est bien documenté notamment dans le cas de la β-glucuronidase, ainsi que dans le cas de la ribonucléase B (Arch. Biochem. Biophys. (1978) 188, 418 ; Advances in Enzymology Meister A. Ed New York (1974) ; Pediat. Res. (1977) 11, 816).It has thus been shown that the glycoproteins recognized by these receptors - present essentially on Kupffer's cells - are rapidly eliminated from the blood circulation by fixation on these cells which metabolize them. This is well documented in particular in the case of β-glucuronidase, as well as in the case of ribonuclease B (Arch. Biochem. Biophys. (1978) 188, 418; Advances in Enzymology Meister A. Ed New York (1974); Pediat. Res. (1977) 11, 816).

De l'ensemble de ces données, il apparaît que la rapide élimination des immunotoxines peut s'expliquer par la reconnaissance des résidus mannose de la chaîne A de ricine par les cellules hépatiques et en particulier les cellules de Kupffer. La propriété des mannanes d'inhiber l'élimination plasmatique rapide des immunotoxines à chaîne A de ricine s'explique en outre aisément par le fait que les mannanes administrés occupent les récepteurs cellulaires des glycoprotéines et s'opposent donc à la reconnaissance par ces récepteurs des motifs polyosidiques portés par la chaîne A de ricine ou 'par tout conjugué la contenant.From all of these data, it appears that the rapid elimination of immunotoxins can be explained by the recognition of mannose residues of the ricin A chain by liver cells and in particular Kupffer cells. The property of mannanes to inhibit the rapid plasma elimination of ricin A-chain immunotoxins is also easily explained by the fact that administered mannanes occupy the cellular receptors of glycoproteins and therefore oppose recognition by these receptors of polysaccharide units carried by the ricin A chain or 'by any conjugate containing it.

La propriété des mannanes d'inhiber l'élimination plasmatique rapide des immunotoxines à chaîne A de ricine s'applique aussi bien, pour les raisons indiquées plus haut, à la chaîne A de ricine non couplée ou à toute molécule hybride, naturelle ou semi-synthétique, contenant la chaîne A de ricine et, en particulier, à toutes les immunotoxines contenant la chaîne A de ricine, quel que soit le type de liaison choisi pour relier l'anticorps à la chaîne A de ricine. Cette propriété des mannanes s'applique plus généralement pour les mêmes raisons à tout immunotoxine, quelle que soit la toxine utilisée pour la produire, si cette toxine ou sous-unité toxique comporte des groupements polysaccharidiques contenant des résidus de mannose, en particulier en position terminale, quel que soit l'anticorps constitutif et quel que soit le type de liaison choisi pour relier l'anticorps à la toxine ou sous-unité toxique.The property of mannanes to inhibit the rapid plasma elimination of ricin A chain immunotoxins applies equally, for the reasons indicated above, to the uncoupled ricin A chain or to any hybrid, natural or semi-molecule synthetic, containing the ricin A chain and, in particular, to all the immunotoxins containing the ricin A chain, whatever the type of binding chosen to link the antibody to the ricin A chain. This property of mannans applies more generally for the same reasons to any immunotoxin, whatever the toxin used to produce it, if this toxin or toxic subunit contains polysaccharide groups containing mannose residues, in particular in the terminal position , whatever the constitutive antibody and whatever the type of binding chosen to bind the antibody to the toxin or toxic subunit.

Les exemples suivants permettent de mieux comprendre l'invention sans en limiter la portée.The following examples provide a better understanding of the invention without limiting its scope.

Exemple 1Example 1

Cet exemple a pour objet de mettre en évidence les modifications de cinétique d'élimination des immunotoxines (ainsi que de celles de leurs éléments constitutifs) en présence ou en absence de mannane.The purpose of this example is to demonstrate the modifications in the kinetics of elimination of the immunotoxins (as well as those of their constituent elements) in the presence or in the absence of mannan.

A - Les modes opératoires suivants ont été utilisés :A - The following procedures were used:

a) Mesure de la cinétique d'élimination de l'immunotoxine IT-T101.a) Measuring the elimination kinetics of the IT-T101 immunotoxin.

Le conjugué appelé IT-T101 est obtenu par réaction entre un anticorps anticellules T humaines (anticorps T101 dirigé contre l'antigène T65) substitué par un groupe disulfure activé et la chaîne A de ricine. La préparation et les propriétés cytotoxiques de ce conjugué ont été décrites dans la demande de brevet français n° 8121836 de la demanderesse. Le conjugué tT-T101 est administré chez le lapin par injection unique dans une veine de l'oreille. La quantité injectée correspond à 1,25 mg d'immunotoxine par kg de poids corporel, soit 0,415 mg/kg exprimé en chaîne A, et 0,835 mg/kg exprimé en anticorps. Les échantillons de sang sont prélevés au cours du temps sur héparine. Les plasmas sont analysés à l'aide d'un test immunoradiométrique ci-après désigné par l'abréviation IRM-1.The conjugate called IT-T101 is obtained by reaction between an antibody against human T cells (antibody T101 directed against the T65 antigen) substituted by an activated disulfide group and the ricin A chain. The preparation and the cytotoxic properties of this conjugate have been described in French patent application No. 8121836 of the applicant. The tT-T101 conjugate is administered to rabbits by a single injection into an ear vein. The amount injected corresponds to 1.25 mg of immunotoxin per kg of body weight, ie 0.415 mg / kg expressed in chain A, and 0.835 mg / kg expressed in antibody. Blood samples are taken over time on heparin. The plasmas are analyzed using an immunoradiometric test below designated by the abbreviation IRM-1.

Cette technique a l'avantage de doser l'immunotoxine sans modification de celle-ci. Ce dosage est réalisé dans des plaques à microtitrations (par exemple : « NUNC-TSP screening system » Poly Labo Block, France) dont le couvercle est muni de pointes hyper-absorbantes qui plongent dans les puits du socle. Ces pointes constituent les phases solides. Des anticorps de brebis antichaîne A de ricine (ci-après désignés sous l'abréviation Acl), purifiés par chromatographie d'affinité, sont adsorbés sur les phases solides. Pour cela, 200 µl d'une solution d'Acl à 10 µg/ml dans le tampon phosphate 20 mM pH 7, NaCi 150 mM sont distribués dans les puits. Les pointes sont mises en contact d'abord avec la solution d'Acl pendant 24 h à 4 °C et ensuite avec du sérum de veau foetal pendant 3 h à 20 °C pour saturer tous les sites de fixation. L'immunoabsorbant saturé est alors mis en contact pendant 3 h à 20 °C avec les échantilons plasmatiques à différentes dilutions ou avec des solutions d'immunotoxine IT-T101 de concentrations connues pour l'établissement de la courbe d'étalonnage. Un lavage est effectué par un tampon phosphate 20 mM pH 7, NaCI 150 mM puis l'immunoabsorbant est mis en contact pendant 2 h à 20 °C avec des anticorps de chèvre anti-IgG de souris purifiés par chromatographie d'affinité et radiomarqués (ci-après désignés par l'abréviation Ac2). Le radiomarquage de l'Ac2 est réalisé avec l'iode 125 en présence de chloramine T selon la méthode de Greenwood et Hunter (Biochem. J., (1963) 89, 114) ; l'activité spécifique des Ac2 radiomarqués est de 5 à 10 µCi/mg. 106 cpm d'Ac2 radiomarqués sont introduits sous 200 ml dans un tampon phosphate 20 mM pH 7, NaCi 150 mM, sérum albumine bovine 0,1 %. Après lavage, dans un tampon phosphate 20 mM pH 7, NaCI 150 mM, les pointes sont détachées et la quantité d'Ac2 liée est mesurée par comptage de la radioactivité. La mesure de la concentration en immunotoxine dans les échantillons à doser se fait par référence à la courbe d'étalonnage réalisée avec l'IT-T101 introduite à différentes concentrations connues.This technique has the advantage of assaying the immunotoxin without modifying it. This assay is carried out in microtiter plates (for example: “NUNC-TSP screening system” Poly Labo Block, France), the cover of which is provided with hyper-absorbent tips which plunge into the wells of the base. These points constitute the solid phases. Ricin antichain A sheep antibodies (hereinafter referred to as Acl), purified by affinity chromatography, are adsorbed on the phases solid. For this, 200 μl of an Acl solution at 10 μg / ml in the 20 mM phosphate buffer pH 7, 150 mM NaCi are distributed in the wells. The tips are brought into contact first with the Acl solution for 24 h at 4 ° C and then with fetal calf serum for 3 h at 20 ° C to saturate all the binding sites. The saturated immunoabsorbent is then brought into contact for 3 h at 20 ° C with the plasma samples at different dilutions or with IT-T101 immunotoxin solutions of known concentrations for establishing the calibration curve. Washing is carried out with 20 mM phosphate buffer, pH 7, 150 mM NaCl, then the immunoabsorbent is brought into contact for 2 h at 20 ° C. with goat anti-mouse IgG antibodies purified by affinity chromatography and radiolabelled ( hereinafter designated by the abbreviation Ac2). Radiolabelling of Ac2 is carried out with iodine 125 in the presence of chloramine T according to the method of Greenwood and Hunter (Biochem. J., (1963) 89, 114); the specific activity of radiolabelled Ac2 is 5 to 10 µCi / mg. 10 6 cpm of radiolabelled Ac2 are introduced under 200 ml in a 20 mM phosphate buffer pH 7, 150 mM NaCl, 0.1% bovine serum albumin. After washing, in a 20 mM phosphate buffer pH 7, 150 mM NaCl, the tips are detached and the amount of bound Ac2 is measured by counting the radioactivity. The measurement of the immunotoxin concentration in the samples to be assayed is made by reference to the calibration curve carried out with the IT-T101 introduced at different known concentrations.

Ce test - par son principe de reconnaissance - permet de mesurer les molécules d'immunotoxine intactes.This test - by its principle of recognition - measures the intact immunotoxin molecules.

De plus, la comparaison des concentrations obtenues dans cet essai avec celles mesurées selon le test d'activité cytotoxique in vitro sur cellules cibles donne des valeurs identiques, ce qui assure que l'immunotoxine dosée par le test IRM-1 correspond à des molécules qui ont conservé leur propriété de cytotoxicité.In addition, the comparison of the concentrations obtained in this test with those measured according to the test of cytotoxic activity in vitro on target cells gives identical values, which ensures that the immunotoxin assayed by the MRI-1 test corresponds to molecules which have retained their cytotoxicity property.

b) Mesure de la cinétique d'élimination de l'anticorps anticellules T humaines (ou anticorps T101).b) Measurement of the kinetics of elimination of the human anti-T cell antibody (or T101 antibody).

Cet anticorps a été préparé et purifié ainsi qu'il a été indiqué dans la demande de brevet français n* 8121836. L'anticorps T101 est injecté chez le lapin par voie intraveineuse à la dose de 0,835 mg/kg. Les prélèvements plasmatiques sont effectués comme précédemment. La concentration en anticorps dans les échantillons est mesurée par dosage radioimmunométrique (IRM-2). Cet essai est réalisé dans les mêmes conditions que le test IRM-1, excepté que la solution Acl est ici une solution à 10 mg/ml d'anticorps de chèvre anti-IgG de souris purifiés par chromatographie d'affinité. L'anticorps Ac2 est identique à celui du test IRM-1. La mesure de la concentration en anticorps T101 dans les échantillons a doser se fait par référence à la courbe d'étalonnage réalisée avec l'anticorps T101 introduit à différentes concentrations connues.This antibody was prepared and purified as indicated in French patent application No. 8121836. The T101 antibody is injected into rabbits intravenously at a dose of 0.835 mg / kg. Plasma samples are taken as before. The antibody concentration in the samples is measured by radioimmunometric assay (MRI-2). This test is carried out under the same conditions as the MRI-1 test, except that the Acl solution is here a 10 mg / ml solution of goat anti-mouse IgG antibodies purified by affinity chromatography. The Ac2 antibody is identical to that of the MRI-1 test. The measurement of the concentration of T101 antibody in the samples to be assayed is made by reference to the calibration curve carried out with the T101 antibody introduced at different known concentrations.

c) Mesure de la cinétique d'élimination de la chaîne A de ricine.c) Measurement of the kinetics of elimination of the ricin A chain.

La chaîne A de ricine a été préparée et purifiée ainsi qu'il a été indiqué dans les brevets français antérieurs n' 78/27838 et addition n' 79/24655. La chaîne A est injectée chez le lapin par voie intraveineuse à la dose de 0,415 mg/kg. Les prélèvements plasmatiques sont effectués comme précédemment. La concentration en chaîne A dans les échantillons est mesurée par dosage immunoradiométrique (IRM-3). Cet essai est réalisé dans les mêmes conditions que le test IRM-1, les anticorps Ad, absorbés sur la phase solide, étant également des anticorps de brebis antichaîne A de ricine purifiés par chromatographie d'affinité et les anticorps Ac2 étant les mêmes anticorps radiomarqués comme décrits dans IRM-1. La mesure de la concentration en chaîne A de ricine dans les échantillons à doser se fait par référence à une courbe d'étalonnage réalisée avec la chaîne A de ricine introduite à différentes concentrations connues.The ricin chain A was prepared and purified as indicated in prior French patents No. 78/27838 and addition No. 79/24655. The A chain is injected into the rabbit intravenously at a dose of 0.415 mg / kg. Plasma samples are taken as before. The concentration of chain A in the samples is measured by immunoradiometric assay (MRI-3). This test is carried out under the same conditions as the MRI-1 test, the antibodies Ad, absorbed on the solid phase, also being sheep antibodies against ricin antichain A purified by affinity chromatography and the antibodies Ac2 being the same radiolabeled antibodies as described in IRM-1. The measurement of the ricin A chain concentration in the samples to be assayed is made by reference to a calibration curve carried out with the ricin A chain introduced at different known concentrations.

Les valeurs des concentrations en immunotoxine, anticorps et chaîne A de ricine dans les plasmas, mesurées par ces trois tests, sont reproductibles, fiables et quantitatives. Le seuil de détection pour ces trois produits est de 1 ng/ml. L'étude de la reproductibilité intra- et inter-essais donne des coefficients de variation inférieurs à 10 % pour les valeurs de concentration comprises dans la gamme de 1 à 200 ng/ml.The values of the immunotoxin, antibody and ricin A chain concentrations in plasmas, measured by these three tests, are reproducible, reliable and quantitative. The detection threshold for these three products is 1 ng / ml. The study of intra- and inter-test reproducibility gives coefficients of variation of less than 10% for the concentration values in the range from 1 to 200 ng / ml.

B - Résultats :

  • Les résultats des expériences effectuées sont représentés sous la forme de courbes présentant en abscisses le temps, exprimé en heures, et, en ordonnées, sur une échelle logarithmique, la concentration du produit mesuré, rapportée en pourcent de la concentration plasmatique théorique au temps zéro. Cette valeur appelée « concentration plasmatique relative » (CPR) est calculée à l'aide de l'expression suivante :
    Figure imgb0001
    Le volume plasmatique est considéré égal à 36 ml/kg de poids corporel de l'animal.
B - Results:
  • The results of the experiments carried out are represented in the form of curves presenting the time on the abscissa, expressed in hours, and, on the ordinate, on a logarithmic scale, the concentration of the measured product, reported as a percentage of the theoretical plasma concentration at time zero. This value called “relative plasma concentration” (CPR) is calculated using the following expression:
    Figure imgb0001
     The plasma volume is considered equal to 36 ml / kg of body weight of the animal.

a) En l'absence de mannane : cinétique d'élimination plasmatique de l'immunotoxine IT-T101, de l'anticorps T101 et de la chaîne A de ricine.a) In the absence of mannan: kinetics of plasma elimination of the IT-T101 immunotoxin, the T101 antibody and the ricin A chain.

La figure 1 montre la courbe d'élimination plasmatique de l'IT-T101 qui présente deux phases (courbe 1). Dans la première phase, le produit disparaît rapidement (environ 97 % en 30 min) ; dans la deuxième phase, la décroissance est plus lente. La première phase d'élimination observée avec l'IT-T101 n'apparaît pas dans la cinétique d'élimination de l'anticorps T101 où seule une phase d'élimination, lente, est enregistrée (courbe 2). Par contre, la cinétique d'élimination plasmatique de la chaîne A non couplée est très comparable à celle de l'IT-T101 : 1 h après l'injection, il ne reste plus dans le plasma que 0,7 % de la dose administrée (courbe 3).Figure 1 shows the plasma elimination curve of IT-T101 which has two phases (curve 1). In the first phase, the product disappears quickly (around 97% in 30 min); in the second phase, the decrease is slower. The first elimination phase observed with IT-T101 does not appear in the elimination kinetics of the T101 antibody where only a slow elimination phase is recorded (curve 2). On the other hand, the kinetics of plasma elimination from the uncoupled A chain is very comparable to that of IT-T101: 1 h after the injection, only 0.7% of the administered dose remains in the plasma. (curve 3).

b) En présence de mannane : cinétique d'élimination plasmatique de l'immunotoxine IT-T101 et de la chaîne A de ricine.b) In the presence of mannan: kinetics of plasma elimination of the immunotoxin IT-T101 and of the ricin A chain.

L'administration du mannane a été effectuée selon le schéma suivant :

  • 10 min avant l'injection de l'IT-T101 ou de la chaîne A, 20 % de la dose finale du polysaccharide sont injectés par voie intraveineuse. Au temps zéro, 40 % de la dose finale du polysaccharide sont injectés par voie intraveineuse en association avec l'lT-T101 ou la chaîne A (0,415 mg de chaîne A/kg). Puis, respectivement aux temps 1,5 h et 5 h, 20 % de la dose finale de polysaccharide sont injectés par voie intraveineuse.
The administration of the mannan was carried out according to the following scheme:
  • 10 min before the injection of IT-T101 or of the A chain, 20% of the final dose of the polysaccharide is injected intravenously. At time zero, 40% of the final dose of the polysaccharide is injected intravenously in combination with lT-T101 or the A chain (0.415 mg of A chain / kg). Then, respectively at times 1.5 h and 5 h, 20% of the final dose of polysaccharide is injected intravenously.

La figure 2 montre la courbe d'élimination plasmatique en fonction du temps de l'IT-T101 injectée par voie intraveineuse en association avec le mannane à la dose totale de 0,416 g/kg (courbe 2). En présence de mannane, la première phase d'élimination - responsable de la disparition de la plus grande partie du produit - est pratiquement abolie, ce qui conduit à un accroissement majeur du taux d'immunotoxine plasmatique active. 15 h après l'injection, la concentration de l'IT-Ti 01 est 100 fois supérieure, quand l'immunotoxine a été associée au mannane, à ce qu'elle est en l'absence de mannane (courbe 1).Figure 2 shows the plasma elimination curve as a function of time of IT-T101 injected intravenously in association with mannan at the total dose of 0.416 g / kg (curve 2). In the presence of mannan, the first phase of elimination - responsible for the disappearance of most of the product - is practically abolished, which leads to a major increase in the level of active plasma immunotoxin. 15 h after the injection, the concentration of IT-Ti 01 is 100 times higher, when the immunotoxin has been combined with mannan, than it is in the absence of mannan (curve 1).

Cet effet d'inhibition de l'élimination plasmatique de l'immunotoxine est dépendant de la dose de mannane. Des doses inférieures de mannane (166 mg/kg et 16,6 mg/kg) produisent des effets plus faibles (courbes 3 et 4).This inhibitory effect on the plasma elimination of the immunotoxin is dependent on the dose of mannan. Lower doses of mannan (166 mg / kg and 16.6 mg / kg) produce weaker effects (curves 3 and 4).

Les propriétés du mannane sont également observées avec la chaîne A non couplée comme le montre la figure 3. Ce point confirme que la rapide disparition de l'immunotoxine est bien imputable à la chaîne A constitutive, en particulier en raison de ses résidus mannosidiques terminaux.The properties of mannan are also observed with the uncoupled A chain as shown in FIG. 3. This point confirms that the rapid disappearance of the immunotoxin is indeed attributable to the constitutive A chain, in particular because of its terminal mannosidic residues.

On constate néanmoins que la première phase d'élimination de la chaîne A n'est pas totalement abolie, contrairement à ce qui se passe avec l'immunotoxine. Ceci confirme l'effet totalement surprenant observé lors de l'administration simultanée de mannane et d'immunotoxine.However, it can be seen that the first phase of elimination of the A chain is not completely abolished, unlike what happens with the immunotoxin. This confirms the completely surprising effect observed during the simultaneous administration of mannan and immunotoxin.

. Exemple 2. Example 2

Afin de montrer la spécificité d'action éventuelle du mannane, la cinétique d'élimination plasmatique de l'immunotoxine IT-T101 a été mesurée en présence d'autres polysaccharides ne possédant pas de résidus mannose en position terminale. C'est le cas des dextrans'TIO, T40 ou T500 (polymères de glucose de poids moléculaires voisins de 10 000, 40 000 et 500 000, respectivement) administrés à la dose totale de 416 mg/kg, du galactane (polymère de galactose) administré à la dose totale de 166 mg/kg de l'asialofétuine (glycoprotéine fortement glycosylée à galactose terminal) administrée à la dose totale de 166 mg/kg.In order to show the specificity of possible action of mannan, the kinetics of plasma elimination of the IT-T101 immunotoxin was measured in the presence of other polysaccharides having no mannose residues in the terminal position. This is the case with dextrans'TIO, T40 or T500 (glucose polymers with molecular weights close to 10,000, 40,000 and 500,000, respectively) administered at the total dose of 416 mg / kg, galactan (galactose polymer ) administered at the total dose of 166 mg / kg of asialofetuin (highly glycosylated glycoprotein with terminal galactose) administered at the total dose of 166 mg / kg.

Les courbes représentées sur la figure 4 montrent que ces polysaccharides sont pratiquement sans action sur la cinétique d'élimination plasmatique de l'immunotoxine IT-T101..The curves shown in FIG. 4 show that these polysaccharides have practically no action on the kinetics of plasma elimination of the IT-T101 immunotoxin.

Exemple 3Example 3

Cet exemple démontre la captation hépatique de la chaîne A de ricine après injection intraveineuse et l'inhibition de cette captation par le mannane.This example demonstrates the hepatic uptake of the ricin A chain after intravenous injection and the inhibition of this uptake by mannan.

La chaîne A radiomarquée à l'iode 125 est injectée chez la souris Charles River France CD1 par voie intraveineuse en l'absence ou en présence de mannane à la dose de 1 g/kg. Aux différents temps de l'expérience, deux animaux sont anesthésiés. La cavité abdominale est ouverte, la veine cave sectionnée, le foie est lavé avec 10 ml d'eau physiologique par injection dans la veine porte. Le foie est prélevé dans sa totalité et la radioactivité est déterminée. Les résultats sont représentés en pourcentage du nombre de cpm fixés sur le foie par rapport au nombre de cpm totaux injectés (figure 5). En l'absence de mannane, la chaîne A de ricine est très précocement et efficacement captée par le foie comme l'indique le pic de radioactivité. Inversement, en présence de mannane, ce pic de radioactivité est pratiquement aboli. Ce résultat confirme que la chaîne A de ricine est piégée par le foie et que le maintien de l'immunotoxine comme de la chaîne A à des taux plasmatiques élevés en présence de mannane est bien dû à l'inhibition de cette capture hépatique.The i-125 radiolabelled A chain is injected into Charles River France CD1 mice intravenously in the absence or presence of mannan at a dose of 1 g / kg. At the different times of the experiment, two animals are anesthetized. The abdominal cavity is open, the vena cava sectioned, the liver is washed with 10 ml of physiological water by injection into the portal vein. The entire liver is removed and the radioactivity is determined. The results are represented as a percentage of the number of cpm fixed on the liver relative to the number of total cpm injected (FIG. 5). In the absence of mannan, the ricin A chain is very early and efficiently picked up by the liver as indicated by the peak of radioactivity. Conversely, in the presence of mannan, this peak of radioactivity is practically abolished. This result confirms that the ricin A chain is trapped by the liver and that the maintenance of the immunotoxin as of the A chain at high plasma levels in the presence of mannan is indeed due to the inhibition of this hepatic capture.

Exemple 4Example 4

Cet exemple démontre l'absence d'effet antagoniste du mannane vis-à-vis de la cytotoxicité sélective de l'immunotoxine IT-T101 in vitro.This example demonstrates the absence of an antagonistic effect of mannan with respect to the selective cytotoxicity of the immunotoxin IT-T101 in vitro.

Dans ces expériences, la cytotoxicité a été évaluée par la mesure de l'incorporation de 14C-leucine par les cellules cibles (cellules CEM) après 24 h d'incubation à 37 °C en présence de concentrations connues de l'immunotoxine étudiée, ou de substances cytotoxiques de référence, en absence ou présence de mannane à la concentration de 10 mg/ml. La technique employée est celle précédemment décrite (J. Biol. Chem. 1984, 259 (15), 9359).In these experiments, the cytotoxicity was evaluated by measuring the incorporation of 14C-leucine by the target cells (CEM cells) after 24 h of incubation at 37 ° C. in the presence of known concentrations of the immunotoxin studied, or of reference cytotoxic substances, in the absence or presence of mannan at a concentration of 10 mg / ml. The technique used is that previously described (J. Biol. Chem. 1984, 259 (15), 9359).

Il a été préalablement vérifié que le mannane n'est pas cytotoxique pour les cellules aux concentrations utilisées. Les résultats de ces expériences sont présentés dans le tableau 1. L'effet cytotoxique est mesuré par la valeur de la concentration molaire (CI50) exprimée en chaîne A qui entraîne une inhibition de l'incorporation du traceur de 50 %.It has been previously verified that the mannan is not cytotoxic for the cells at the concentrations used. The results of these experiments are presented in Table 1. The cytotoxic effect is measured by the value of the molar concentration (IC50) expressed in chain A which results in an inhibition of the incorporation of the tracer by 50%.

L'immunotoxine seule ou sous sa forme activée par le chlorure d'ammonium conserve pleinement son activité. De la même façon, la toxicité intrinsèque de la chaîne A n'est pas modifiée. Ainsi, en . présence de mannane, les propriétés caractéristiques de cytotoxicité de l'immunotoxine ne sont pas altérées.

Figure imgb0002
The immunotoxin alone or in its form activated by ammonium chloride fully retains its activity. Likewise, the intrinsic toxicity of the A chain is not modified. So in. presence of mannan, the characteristic cytotoxicity properties of the immunotoxin are not altered.
Figure imgb0002

Exemple 5Example 5

Toxicité de l'immunotoxine IT-T101 injectée en association avec le mannane chez la souris.Toxicity of the IT-T101 immunotoxin injected in combination with mannan in mice.

Il était important de vérifier quel était l'impact toxicologique global sur l'animal entier de l'association immunotoxine plus mannane. Pour cela, nous avons déterminé la dose létale 50 % de l'immunotoxine antimélanome (IT-HM) administrée par voie intraveineuse chez la souris Charles River France CD1 en l'absence ou avec coadministration de 10 mg de mannane par souris par voie intraveineuse. La préparation et les propriétés cytotoxiques de ce conjugué antimélanome (IT-HM) ont été décrites dans la demande de brevet français n° 81/07596.It was important to verify the overall toxicological impact on the whole animal of the immunotoxin plus mannan combination. For this, we determined the lethal dose 50% of the immunotoxin antimelanoma (IT-HM) administered intravenously in Charles River France CD1 mice in the absence or with coadministration of 10 mg of mannan by mouse intravenously. The preparation and cytotoxic properties of this antimelanoma conjugate (IT-HM) have been described in French patent application No. 81/07596.

Les valeurs trouvées sont indiquées dans le tableau II.

Figure imgb0003
The values found are shown in Table II.
Figure imgb0003

Ces résultats montrent une légère augmentation de toxicité de l'immunotoxine lorsqu'elle est administrée en même temps que le mannane. Cet accroissement de toxicité par un facteur de seulement 4 ne constitue pas une limitation à l'emploi in vivo de mannane compte tenu des effets très importants vis-à-vis du maintien de la concentration plasmatique des immunotoxines in vivo, comme cela été démontré ci- dessus.These results show a slight increase in the toxicity of the immunotoxin when administered at the same time as mannan. This increase in toxicity by a factor of only 4 does not constitute a limitation to the in vivo use of mannan taking into account the very significant effects with respect to the maintenance of the plasma concentration of immunotoxins in vivo, as has been demonstrated above. - above.

Exemple 6Example 6

Cet exemple a pour but de mettre en évidence l'effet du mannane sur l'action d'une immunotoxine dans un essai « in vivo ».The purpose of this example is to demonstrate the effect of mannan on the action of an immunotoxin in an "in vivo" test.

L'expérimentation a été effectuée chez des souris BL 1.1 (Thy 1.2 négatives) [International Journal of Cancer 24, 168-177, (1979)]. L'immunotoxine utilisée est le conjugué associant par une liaison disulfure l'anticorps anti-Thy 1.2 (anticorps AT15E) et la chaîne A de la Ricine et préparé selon le procédé décrit dans les demandes de brevets français au nom de la demanderesse et citées précédemment.The experiment was carried out in BL 1.1 (Thy 1.2 negative) mice [International Journal of Cancer 24, 168-177, (1979)]. The immunotoxin used is the conjugate combining via a disulfide bond the anti-Thy 1.2 antibody (AT15E antibody) and the A chain of Ricin and prepared according to the process described in the French patent applications in the name of the applicant and cited above.

Le protocole suivant a été utilisé.The following protocol was used.

Des groupes de 10 souris BL 1.1 reçoivent par injection intraveineuse le jour 0,5 x 104 cellules T2 (cellules Thy 1.2 positives de lymphome murin) et sont randomisés avant traitement.Groups of 10 BL 1.1 mice receive 0.5 x 10 4 T2 cells per day intravenous injection (Thy 1.2 positive murine lymphoma cells) and are randomized before treatment.

Le traitement est effectué par voie intraveineuse le jour 1 :

  • 1 lot reçoit 10 µg/souris du conjugué anticorps AT15E-chaîne A de ricine seul.
  • 1 autre lot reçoit la même quantité du même conjugué mélangé avec 10 mg de mannane.
Treatment is carried out intravenously on day 1:
  • 1 batch receives 10 μg / mouse of the AT15E antibody-ricin chain conjugate alone.
  • 1 other batch receives the same quantity of the same conjugate mixed with 10 mg of mannan.

-De plus 4 lots témoins reçoivent respectivement :

  • le milieu de culture RPMI (milieu utilisé pour la culture des cellules T2) ;
  • le mannane seul (10 mg/souris) ;
  • la chaîne A de ricine (10 µg) et le mannane (10 mg) ;
  • la chaîne A de ricine (10 µg), l'anticorps AT15E (30 lig) et le mannane (10 mg).
-More than 4 control lots receive respectively:
  • RPMI culture medium (medium used for the culture of T2 cells);
  • mannan alone (10 mg / mouse);
  • ricin chain A (10 µg) and mannan (10 mg);
  • the ricin chain A (10 µg), the antibody AT15E (30 li g) and the mannan (10 mg).

Les animaux sont observés pendant 50 jours et la mortalité est notée.The animals are observed for 50 days and mortality is noted.

La figure 6 donne le pourcentage d'animaux survivants en fonction du temps écoulé après le traitement.Figure 6 gives the percentage of surviving animals as a function of the time elapsed after treatment.

La courbe 1 concerne les animaux ayant reçu l'immunotoxine seule et la courbe 2 les animaux ayant reçu l'association immunotoxine plus mannane.Curve 1 concerns the animals which received the immunotoxin alone and curve 2 the animals which received the immunotoxin plus mannan combination.

Ainsi qu'on peut le constater, l'association immunotoxine-mannane a donné 90 % de survivants 50 jours après le traitement alors que l'administration de l'immunotoxine seule n'a donné que 30 % d'animaux survivants après 50 jours.

  • De plus, sur les lots témoins (non représentés sur la figure 6), on a observé au jour 50 :
  • 0 % de survivants pour les animaux traités par RPMI et par chaîne A plus mannane ;
  • 10 % de survivants pour les animaux ayant reçu le mannane seul ;
  • 20 % de survivants pour les animaux traités par chaîne A de ricine + anticorps + mannane.
As can be seen, the immunotoxin-mannan association gave 90% of survivors 50 days after the treatment whereas the administration of immunotoxin alone gave only 30% of animals surviving after 50 days.
  • In addition, on the control batches (not shown in FIG. 6), on day 50, we observed:
  • 0% survivors for animals treated with RPMI and with chain A plus mannan;
  • 10% survivors for animals that received the mannan alone;
  • 20% of survivors for animals treated with ricin A chain + antibody + mannan.

Ces résultats montrent l'efficacité de l'association immunotoxine-mannane aussi bien vis-à-vis de l'immunotoxine utilisée seule que vis-à-vis des témoins.These results show the effectiveness of the immunotoxin-mannan association both vis-à-vis the immunotoxin used alone and vis-à-vis the controls.

On peut donc utiliser en tant que médicaments en thérapeutique humaine l'association constituée par une immunotoxine et du mannane. Cette association peut être utilisée pour le traitement des affections, cancéreuses ou non, dont les cellules cibles seraient reconnues par l'anticorps utilisé pour la préparation de l'immunotoxine.It is therefore possible to use, as medicaments in human therapy, the combination constituted by an immunotoxin and mannan. This combination can be used for the treatment of conditions, cancerous or not, whose target cells are recognized by the antibody used for the preparation of the immunotoxin.

Visant à éliminer la totalité des cellules cibles, le traitement devra être effectué avec une dose suffisante d'immunotoxine associée avec une quantité de mannane pouvant varier de 10 mg à 1 g/kg à chaque administration d'immunotoxine. Les modalités optimales d'administration des constituants de l'association ainsi que la durée du traitement devront être déterminées dans chaque cas en fonction du sujet et de la nature de l'affection à traiter.Aiming to eliminate all of the target cells, the treatment should be carried out with a sufficient dose of immunotoxin associated with an amount of mannan which can vary from 10 mg to 1 g / kg each time the immunotoxin is administered. The optimal methods of administration of the constituents of the association as well as the duration of treatment must be determined in each case depending on the subject and the nature of the condition to be treated.

Les nouveaux médicaments selon l'invention sont conditionnés pour être utilisés par voie injectable et, de préférence, par voie intraveineuse. De préférence, les constituants de l'association seront conservés séparés et pourront être mélangés en cas de besoin seulement au moment de l'emploi, dans la seringue ou le solvant de perfusion.The new drugs according to the invention are packaged for use by the injectable route and, preferably, by the intravenous route. Preferably, the constituents of the combination will be kept separate and may be mixed if necessary only at the time of use, in the syringe or the perfusion solvent.

Claims (4)

1. Drugs packaged for administration by injection, characterized in that they comprise an association of at least one immunotoxin obtained from a natural, semi-synthetic or synthetic toxin, toxic sub-unit or fragment of toxic sub-unit comprising polysaccharide groups containing mannose residues, in particular in the terminal position, irrespective of the constituent antibody and irrespective of the type of bond chosen to join the antibody to the toxin, toxic sub-unit or toxic fragment, and at least a polyoside or polysaccharide carbohydrate polymer which has an average molecular weight greater than 1 000 comprising from 20 to 100 % of mannose residues, irrespective of the type of linkage joining these mannose residues to one another or to other sugars.
2. Drugs according to claim 1, characterized in that the immunotoxin is the immunotoxin anti-T65 and the mannose-containing polymer is a mannane.
3. Drugs according to any one of claims 1 or 2, characterized in that they are packaged for intravenous injection.
4. Drugs according to any one of claims 1 to 3, characterized in that the immunotoxin and the mannose-containing polymer are packaged separately.
EP85402319A 1984-11-29 1985-11-27 Medicament containing as a mixture at least one immunotoxin and at least one polymer containing mannose Expired - Lifetime EP0186551B1 (en)

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US4946675A (en) * 1987-05-27 1990-08-07 Xoma Corporation Hepatic blocking agents
GB8809616D0 (en) * 1988-04-22 1988-05-25 Cancer Res Campaign Tech Further improvements relating to drug delivery systems
US5632990A (en) * 1988-04-22 1997-05-27 Cancer Research Campaign Tech. Ltd. Treatment for tumors comprising conjugated antibody A5B7 and a prodrug
US5112607A (en) * 1991-06-05 1992-05-12 The United States Of America As Represented By The Secretary Of The Army Potentiation of immunotoxin action by Brefeldin A
FR2802536B1 (en) * 1999-11-23 2003-06-13 Chru Lille SYNTHESIS OLIGOMANNOSIDES, THEIR PREPARATION AND THEIR USE FOR THE DETECTION OF ANTIBODIES AND THE PREVENTION OF INFECTIONS
WO2006121803A1 (en) 2005-05-05 2006-11-16 Sensient Flavors Inc. Production of beta-glucans and mannans
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US20120177567A1 (en) * 2010-08-11 2012-07-12 National Institutes Of Health (Nih), U.S. Dept. Of Health And Human Services (Dhhs), U.S. Government Methods of Treating Pediatric Acute Lymphoblastic Leukemia with an Anti-CD22 Immunotoxin

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